The long-term goal of this project is to understand the mechanisms regulating proliferation and differentiation of spermatogonial stem cells in the human and monkey testis. Present knowledge of the mechanisms regulating the initiation of primate spermatogonial self-renewal and differentiation is very limited. We know there are two types of undifferentiated spermatogonia (Adark, Apale), and the differentiation of Apale spermatogonia into B spermatogonia is considered to be the initiating step in spermatogenesis in these species. Three Specific Aims will be addressed: Aim 1 will examine the existence of clonogenic patterns of germ cell expansion, and the impact of paracrine factors in adult testicular tissue will be investigated;Aim 2 will determine the signals responsible for the differentiation of Apale spermatogonia to B spermatogonia;and Aim 3 will determine the potential of xenografting to initiate or restore spermatogonial differentiation, meiotic progression and androgen production in human testicular tissue, in which spermatogenesis has been compromised. These aims will be achieved using organ culture of human and monkey testicular tissue over periods of 72 h and xenografts of testicular tissue into immunodeficient nude mice. These approaches allow us to expose limited amounts of primate testicular tissue to a variety of different conditions by modifying the in vitro environment and applying various treatments to the grafted recipients. We will use a newly developed approach to detect proliferating spermatogonial clones in whole mounts of testis tissue. Dual fluorescence labeling of bromodeoxyuridine and acrosin in conjunction with confocal microscopy allows the description of the clonogenic and spatial arrangement of proliferating spermatogonia at specific stages of the seminiferous epithelial cycle. Crosssections of resin and paraffin-embedded cultured testicular fragments and dissected grafts will be labeled by the same approach. Quantitative morphometric analysis using both, the optical disector and geometric approaches, will be used to determine changes in spermatogonial cell types, numbers and proliferation indices. Therefore, the proposed studies of human and monkey should lead to fundamental advances in our understanding of the control of spermatogenesis in man. This will have clinical relevance because our findings may lead to new concepts in understanding and treating male infertility, and to the development of new strategies for male contraception and for preserving and restoring fertility in patients undergoing gonadotoxic oncological treatments.